Process for fixation of calcification-resistant biological tissue

ABSTRACT

A process is provided for non-glutaraldehyde fixation of an organ or prosthesis to be implanted in a mammal. The process produces stable fixation of the tissue by forming amide linkages within and between the molecules of the tissue and employs a coupling agent, such as EDC, in combination with a coupling enhancer, such as Sulfo-NHS; diamine and/or dicarboxyl cross-linking agents are optionally included. In addition to fixing the tissue, the process prevents or retards calcification and results in a nontoxic product that does not cause inflammation.

This application is a 371 of PCT/US95/02077 filed Feb. 15, 1995 and acontinuation-in-part of our earlier U.S. application Ser. No.08/198,145, filed Feb. 17, 1094, now U.S. Pat. No. 5,447,536 thedisclosure of which is incorporated herein by reference.

The present invention relates to a process for fixing human or animaltissue prior to implantation into humans or animals, and moreparticularly to a fixation process that forms links within and betweenthe proteinaceous molecules of the tissue by covalently binding thereactive amine groups and/or the reactive carboxyl groups on the tissueeither directly in the presence of a coupling agent and preferably of acoupling enhancer, or through bridges formed by one or morecross-linking agent(s) in the presence of a coupling agent andpreferably of a coupling enhancer.

BACKGROUND OF THE INVENTION

The surgical implantation of prosthetic devices (prostheses) into humansand other animals has been carried out with increasing frequency. Suchprostheses include, by way of illustration only, heart valves, vasculargrafts, urinary bladders, left ventricular-assist devices, hips, breastimplants, tendons, and the like. The prosthesis can be entirely orpartially made of biological tissue(s) from humans or from animals. Toprevent degeneration and/or foreign body reactions, the bioprosthetictissue must be stabilized before implantation in a human or in ananimal. The stabilization process, known by those skilled in the art asfixation, consists of blocking the reactive moieties of the tissue.After it was found in 1968 that collagen, a major component ofbioprostheses, was stabilized by aldehydes Nimni et al., J. Biol. Chem.,243:1457-1466 (1968)!, that, of various aldehydes tested, glutaraldehydebest retarded degeneration of implanted heart valves, and thatglutaraldehyde-fixed heart valves were minimally thrombogenic and hadexcellent biophysical and hemodynamic properties Strawich, et al.,Biomat. Med. Dev. Art. Org., 3:309-318 (1975)!, the process ofglutaraldehyde-fixation has been and continues to be applied to mostvarieties of experimental and clinical bioprostheses. This process offixation with glutaraldehyde consists of blocking the reactive amines ofthe tissue through formation of an aldehyde-amine bond known by theskilled in the art as a Schiff-base.

Of all glutaraldehyde-fixed bioprostheses, the heart valve has been oneof the most widely studied, and its clinical application and pathologyare well documented Schoen et al., Cardiovascular Pathology, 1:29-52(1992)!. Heart valve bioprostheses are generally fabricated either fromglutaraldehyde-fixed porcine aortic or pulmonic valves or fromglutaraldehyde-fixed bovine pericardium, which may be sewn, although notnecessarily, onto a cloth-covered metallic or polymeric stent and sewingring. These bioprostheses may be preferred over the mechanical heartvalve prostheses (which typically are composed of rigid materials suchas polymers, pyrocarbons and metals, and employ one or more occluderswhich respond passively with changes in intracardial pressure or flow)because of certain significant clinical advantages. For example, heartvalve bioprostheses do not require permanent anticoagulation therapy,while mechanical heart valves do. Also, should a bioprosthesis fail, ittypically first exhibits a gradual deterioration which can extend over aperiod of months, or even years, while a mechanical heart valve mayoccasionally undergo catastrophic failure. On the other hand,glutaraldehyde-fixed heart valve bioprostheses are generally lessdurable than mechanical heart valves mostly because they calcify.

Calcification has been recognized for more than 20 years as the maincause of failure of most bioprostheses. For example, more than 50percent of heart valve bioprostheses fail within 10 years ofimplantation because of the cuspal tears and stenosis that result fromcalcification, which failure occurs substantially more rapidly inchildren than in adults Schoen et al., Cardiovascular Pathology, 1:29-52(1992)!. Although the pathogenesis of heart valve calcification (whichinvolves not only the donor tissue, but also host factors such as bloodcomponents, and the stress to which the valve is submitted whenimplanted) is as yet not completely understood, glutaraldehyde has beenidentified as an important contributory factor Gong, et al., Eur J.Cardio-Thorac. Surg. 5:288-293 (1991)!. Multiple approaches to eradicatecalcification of glutaraldehyde-fixed bioprostheses have been taken.

The techniques resulting from these efforts may be broadly divided intotwo categories: those involving the treatment of glutaraldehyde-fixedtissue with compounds that prevent calcification, and those involvingthe fixation of tissue with processes that do not induce calcification.The former category of techniques includes, but is not limited to,treatment with anticalcification compounds, such as detergents orsurfactants, diphosphonates, amino acids such as glutamic acid,amino-substituted aliphatic carboxylic acids such as AOA, sulfatedpolysaccharides, trivalent cations such as salts of iron or aluminum,elastomeric polymers, and solutions of phosphate esters, quaternaryammonium salts or sulfated aliphatic alcohols Schoen et al,Cardiovascular Pathology, 1:29-52 (1992); Girardot et al., InternationalJournal of Artificial Organs, 17:127-133 (1994)!. The latter category oftechniques includes, but is not limited to, fixation by photo-oxidationMoore, et al., J. Biomed. Mater. Res., 28:611-618 (1994)!, by treatmentwith polyglycidal ethers Imamura, et al., Jpn. J. Artif. Organs,17:1101-1103 (1988)!or with acyl-azide Petite, et al., J. Biomed. Mater.Res., 24:179-187 (1990)!.

Because of the high incidence of calcification-induced heart valvefailure and the severe clinical implications associated with this typeof failure, which include reoperation, most studies onglutaraldehyde-fixed heart valves have been devoted to the pathogenesisof calcification. However, other problems have been more recentlyidentified, which may also decrease the durability ofglutaraldehyde-fixed bioprostheses. These additional problems are mostlydue to the relative unstability of the Schiff-base formed between thealdehyde and the amine of the tissue and the subsequent slow release oftoxic glutaraldehyde from the tissue. They include low-grade cytotoxiceffects which prevent, for example, the covering of the implantedbioprosthetic tissue by antithrombogenic endothelial cells, low-gradeimmunological reaction by the host and slow degeneration of thebioprosthesis. Although less drastic than calcification-induced failure,this complex glutaraldehyde-related symptomatology is clinicallyimportant, and it can be fully eradicated only if the fixation methoddoes not include glutaraldehyde.

It is therefore an object of this invention to provide a fixation methodthat does not utilize glutaraldehyde, which method is suitable forbioprosthetic tissues to be implanted in humans or in animals.

It is a further object of this invention to provide a fixation processfor biological tissues to be used in bioprostheses, which processresults in stable fixation of the tissue by forming amide linkageswithin and between the molecules of the tissue.

It is also an object of this invention to provide a fixation process forbiological tissues that results in tissues which resist calcification,thus increasing the durability of the bioprosthesis when implanted inhumans or in animals.

It is yet another object of the invention to provide a fixation processfor bioprosthetic tissues that results in tissues which arebiocompatible and do not induce inflammatory responses or toxicreactions when implanted in humans or in animals.

SUMMARY OF THE INVENTION

The fixation process described herein is a cross-linking process thatrelies on the availability of free reactive carboxyl and free reactiveamine moieties on the proteins contained on and within the bioprosthetictissue, which moieties are capable of being linked together throughstable covalent amide bonds in the presence of a coupling agent,preferably with a coupling enhancer, either directly or through bridgesformed by amine and/or carboxyl containing cross-linking agent(s).

In one embodiment, the coupling agent, preferably with a couplingenhancer, is used in the absence of cross-linking agents to promoteamide binding between reactive carboxyl moieties and reactive aminemoieties existing on the tissue. This embodiment should provide adequatefixation for tissues where reactive amines and reactive carboxyls arepresent on such tissues in locations close enough to be directly linkedtogether without any intermediary cross-linking agent.

Where the reactive amine and the reactive carboxyl moieties are toodistant to be attached directly to each other, adequate cross-linking oftissues is attained through cross-linking agents. One such preferredembodiment uses a coupling agent, preferably with a coupling enhancer,in the presence of one or more cross-linking agent(s). When a pluralityof cross-linking agents are used, bridges of various lengths are formedby covalently binding agents to each other, with the extremities of thebridges being attached to the tissue. In this preferred embodiment,reactive moieties located close to each other on the tissue may alsobind directly.

The particular desired physical properties of the bioprosthetic tissuemay also determine which embodiment is employed because the length ofthe links between the molecules of the tissue will have an effect on thephysical properties of the resultant bioprosthetic tissue. For example,for heart valve bioprostheses, where hemodynamic function is related tothe flexibility of the leaflets, a cross-linking process that producesleaflets that are soft and pliable is preferred over one which producesmore rigid leaflets.

The amide bonds formed with this process are more stable than theSchiff-bases formed with the glutaraldehyde process commonly used to fixbiological tissues, and the resultant tissue is as cross-linked and moreresistant to calcification than glutaraldehyde-fixed bioprosthetictissue. In addition, it is not toxic, biocompatible, and does not induceinflammatory responses by the host. The proposed process thus providestissues that are at least as suitable for implantation in humans or inanimals as, and more durable than, glutaraldehyde-fixed tissues.

As used herein, the term "bioprosthetic tissue" is meant to include anyorgan or tissue which is derived in whole or in part from a human or ananimal, or which is made from other organic tissue, and which is to beimplanted by itself or as part of a bioprosthesis, in a human or in ananimal. Thus, the term generally includes bioprosthetic tissue such ashearts, heart valves and other heart components, pericardium, vasculargrafts, urinary tract and bladder components, tendons, bowel, softtissues in general, such as skin, collagen and the like. Although theprosthetic tissue will very often be one which is made from naturaltissues, including but not limited to bovine, ovine, porcine andpossibly even human tissue, other natural materials, well known to thosehaving ordinary skill in this art, also can be used.

The fixation method described herein consists of stabilizing thebioprosthetic tissue by binding a reactive amine or carboxyl moiety ofthe tissue either to another reactive moiety (carboxyl or amine) on thetissue or to one on a cross-linking agent, in such a manner as to leavefew or no active moieties on or within the tissue.

The term "cross-linking", as used herein, refers to the fixation ofbioprosthetic tissue that results from the formation of links of variouslengths within and between the molecules of the tissue, such linksresulting from amide bond formation either (a) between two reactivemoieties of the tissue, thus forming short links within and between themolecules of the tissue, or (b) between reactive moieties on the tissueand each of the respective extremities of bridges formed by one or morecovalently bound cross-linking agent(s), thus forming longer linkswithin and between the molecules of the tissue.

The term "cross-linking agent", as used herein, describes a compoundcontaining at each of its extremities free active amines and/or freeactive carboxyls, which moieties are capable of forming amide bonds withfree active moieties that are located either on other cross-linkingagent(s), thus forming chains of one or more cross-linking agent(s)either on or within the tissue, and thereby linking the free activemoieties of the tissue by attachment to an extremity of such across-linking chain.

One or more cross-linking agents may be used, and preferably, at leasttwo different agents are used. Each cross-linking agent has at least tworeactive moieties which are preferably either carboxyls only or aminesonly. It is preferably a straight-chained or branched compound fromabout 4 to about 24 atoms in length, and most preferably from about 6 toabout 8 atoms in length, with preferably one reactive moiety located ateach extremity, but it can also be a cyclic compound, with the reactivemoieties appropriately located on the ring. When acyclic, difunctionalcompounds are used, the reactive moieties are preferably separated by atleast 4 carbon atoms and more preferably by at least about 6 carbonatoms. Each cross-linking agent may also be appropriately substituted,if desired. They are preferably straight chain alkanes having thereactive moieties at each extremity of the chain, and preferredcross-linking agents include, but are not limited to, suberic acid,adipic acid, terephthalic acid, 1,3,5-benzene tricarboxylic acid,1,6-hexane diamine, 1,7-heptane diamine, triaminobenzoic acid and2,4,6-triaminobenzene.

The concentration of each cross-linking agent can vary and depends onits efficacy to form amide bonds with the bioprosthesis and with theother cross-linking agent(s) used for fixation. In certain preferredembodiments, concentrations ranging from about 5 mM (millimolar) toabout 20 mM are used; however, one skilled in the art can readilydetermine the appropriate concentration for each cross-linking agent.

The terms "coupling agent" and "coupling enhancer", as used herein,refer to reagents that respectively promote and enhance the formation ofamide bonds. These bonds may be formed between a reactive amine and areactive carboxyl on the tissue (thus linking two such closely locatedreactive groups), between a reactive amine on one cross-linking agentand a reactive carboxyl on another cross-linking agent (thus formingchains of various lengths between cross-linking agents), and between tworeactive amines or carboxyls located at the extremities of such across-linking bridge and the reactive carboxyl or amine moieties locatedon and within the tissue (thus forming links of various lengths withinand between the molecules of the bioprosthetic tissue). Those of skillin the peptide synthesis and related art will be familiar with suchreagents, e.g. water-soluble carbodiimides and succinimides.

The coupling agent used in the preferred embodiments is1-ethyl-3(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC),although other suitable coupling agents such as N-hydroxysuccinimide canalso be used. The coupling enhancer used in the embodiment where EDC isused as the coupling agent is N-hydroxysulfosuccinimide (sulfo-NHS),although other suitable coupling enhancers can also be used. Theconcentration of the coupling agent and of the coupling enhancer canvary. However, appropriate concentrations are readily determinable bythose of skill in the art. Preferably, the coupling agent is used in aconcentration between about 10 mM and 500 mM and more preferably atabout 100 mM or less. The coupling enhancer is preferably employed atbetween 0.5 mM and about 50 mM and more preferably at about 10 mM orless.

The cross-linking agents, the coupling agent and the coupling enhanceras well as their reaction products should be preferably water-soluble.They should be selected to be such as to maximize fixation andoptimizing cross-linking of the tissue while minimizing the risks ofdamage to the prosthetic tissue during the fixation process, and oftoxicity, inflammation, calcification, etc, after implantation. Allsolutions used for cross-linking are preferably filtered before usethrough 0.45 μm or less filters to minimize risks of contamination.

Reaction conditions for the cross-linking of the prosthesis may vary,depending on the cross-linking, coupling and enhancing agents employed.In general, the cross-linking process is carried out in an aqueousbuffer selected from among those well known to those of ordinary skillin this art as to provide the most efficacious cross-linking reaction,while minimizing risks of calcification. Examples of suitable buffersinclude, but are not limited to,N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES) and3-(N-morpholino)propanesulfonic acid (MOPS), and the like.

The pH and concentration of the buffered solution also can vary, againdepending upon the cross-linking, coupling and enhancing agentsemployed. In preferred embodiments, the buffer concentration and pH arechosen to provide the most effective cross-linking reaction while beingthe least harmful to the prosthesis. For example, with EDC as thecoupling agent and sulfo-NHS as the coupling enhancer, the pH of thecross-linking reaction is about 6.0 to about 7.4. The temperature of thereaction is maintained between about 40° C. and 0° C.; preferably, thereaction is carried out between 21° and 25° C.

Typically, the fresh prosthetic tissue to be fixed by the cross-linkingmethod described in the present invention is kept on ice until it isrinsed several times in ice-cold 0.85% saline or other solutions knownby those of skill in the art, preferably immediately after and no longerthan 48 hours after being excised from the donor animal. If additionalstorage time is needed, the rinsed tissue is then stored, but not longerthan 24 hours, in an appropriate buffer as described further below, at alow temperature, such as about 4° C.

The bioprosthetic tissue is then cross-linked in one or more consecutivesteps by incubation in the presence of a coupling agent, preferably witha coupling enhancer, either in the presence or in the absence of one ormore cross-linking agents. When more than one cross-linking agent isused together with the coupling and enhancing agents, they can be usedeither in a single step or, preferably, sequentially in severalconsecutive steps.

In a preferred embodiment where a dicarboxylic acid and a diamine areused as cross-linking agents, the two agents are used alternately in athree-step process. As such, either the dicarboxylic acid or the diamineis used in the first step, the alternate agent is used in the secondstep, and the first agent is used again in the third step. At the end ofthis cross-linking process, the free active moieties of the tissue areeither linked together directly, or they are connected together throughbridges composed of between 1 and 5 links alternating between thedicarboxylic acid and the diamine. For illustration, in the preferredembodiment where the diamine is used during the first and third steps,and the diacid is used during the second step, the one-link bridgesresult from attachment of each of two reactive amines of a diamine, oreach of two reactive carboxyls of a diacid to, respectively, tworeactive carboxyl moieties or two reactive amine moieties on the tissue;the five-link bridges result from anchoring one reactive amine from eachof two diamine molecules to a reactive carboxyl on the tissue, the otheramine of each of the two molecules remaining free for further reaction(first step). Each of the two free amines then binds to one reactivecarboxyl group on two different diacid molecules, leaving one carboxylgroup on each of the two diacid molecules free for further reaction(second step). Finally, each of the two free reactive carboxyl moietieson the diacid molecules binds to a reactive amine of a diamine molecule(third step), thus forming the 5-link bridge between two moieties on thetissue.

For each step of the cross-linking process, the time of incubationgenerally depends upon the nature and concentration of thecross-linking, coupling and enhancer agents used, and upon thecross-linking conditions, such as pH and temperature. For instance, anincubation time for each step from about 3 hours to about 48 hours ispreferably employed when EDC and sulfo-NHS are used as the coupling andenhancer agents, respectively. After each step, the tissue is rinsed orwashed in aqueous buffer to remove the non-reacted reagents and theirby-products. At the end of the cross-linking process, the tissue is keptuntil further use in a sterile buffered solution. Appropriate rinsingand buffer solutions are used as previously described and as understoodby those of skill in this art.

The present invention is further described by the examples that follow.Because different bioprosthetic tissues prefer different types ofcross-links depending on the nature of their structure and of theirintended use, the examples describe cross-linking processes that vary interms of the presence or absence of cross-linking agents, of the typesof cross-linking agents, and of the number of steps, thus resulting incross-links that vary in either their nature and/or their complexity.The examples are not to be construed as limiting in any way either thespirit or the scope of the present invention.

The coupling agent used in the examples is 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and the coupling enhanceris N-hydroxy-sulfosuccinimide (sulfo-NHS). They are commerciallyavailable from Sigma and Pierce. The cross-linking agents are1,6-hexane-diamine (DIA), a C-6 straight-chain aliphatic agent with anamine group at each end of the chain, and suberic acid (SUA), a C-8straight-chain aliphatic agent with a carboxyl group at each end of thechain, or 1,3,5-benzenetricarboxylic acid (BCA), an agent with 3reactive carboxyl groups on the benzene ring, are readily obtainablefrom Aldrich. All agents are solubilized in 10 mMHEPES buffer containing0.85% of sodium chloride, pH 6.5 (HEPES buffer). Their concentrationsare expressed as mM (number of millimoles of chemical for each liter ofsolution), or as % (grams per 100 ml of solution). The temperatures arein ° C. (degrees Celsius).

EXAMPLE 1

A process embodying one feature of the present invention is illustratedby the use of EDC and sulfo-NHS in the absence of any cross-linkingagent, thus cross-linking the biological tissue by forming only shortlinks between free active carboxyls and free amines that are locatedclose to each other on the tissue.

1. Preparation of heart valve tissue

Leaflets and 1×1 cm aortic wall coupons were dissected from freshporcine hearts kept on ice. The samples were then rinsed 6 times for 5minutes each time in ice-cold saline to remove red blood cells and otherdebris, and stored overnight at 4° C. in HEPES buffer, pH 6.5. Thesamples used for Examples 1-4 were randomly selected from this originalpool. Samples to be used as "fresh" controls, also selected from thispool, were kept until testing at 4° C. in HEPES buffer containing 20% ofisopropanol. In addition, leaflets and wall coupons dissected fromglutaraldehyde-fixed valves provided by the Heart Valve Division ofMedtronic, Inc., also kept at 4° C. in HEPES buffer containing 20% ofisopropanol, represented the "standard" control condition.

2. Cross-linking of porcine aortic valve

Samples randomly selected from the original pool were incubated threetimes for 48 hours at room temperature in HEPES buffer containing 50 mMof EDC and 2.5 mM of sulfo-NHS, thus forming short links directlybetween free active carboxyl and amines of the tissue. At the end of thecross-linking process, the samples were placed in HEPES buffercontaining 20% of isopropanol.

EXAMPLE 2

Fixation is carried out employing a 3-step cross-linking process thatuses EDC and sulfo-NHS in the presence of SUA in the first step, DIA inthe second step, and SUA again in the third step. Samples randomlyselected from the original pool described in Example 1 were cross-linkedand stored as in Example 1, except that 10 mM SUA, 15 mM DIA and 10 mMSUA were added to EDC and sulfo-NHS during the first, second and thirdsteps of the cross-linking reactions, respectively. As a result, thereare formed the following types of links of various lengths between thefree active moieties of the tissue: direct links, one-link bridges madeof either SUA or DIA, two-link bridges made of the two cross-linkingagents covalently bound to each other, three-link bridges made of chainsformed by one molecule of SUA, one molecule of DIA, and one molecule ofSUA covalently bound together, four-link bridges composed as thethree-link chains with further addition of one molecule of DIA, andfive-link bridges composed as the four-link bridges with furtheraddition of one molecule of SUA. The two extremities of each bridge areconnected via amide bonds with one free moiety on the tissue.

EXAMPLE 3

A 3-step cross-linking process is employed that is similar to theprocess used in Example 2, but which reverses the order of cross-linkingagents used in the three sequential steps. As such, 15 mM DIA was usedin the first step, 10 mM SUA in the second step, and 15 mM DIA again inthe third step, thus forming direct links, one-link bridges made ofeither DIA or SUA with each of the two ends covalently boundrespectively to an amine or a carboxyl group on the tissue, and two-,three-, four- and five-link bridges formed respectively by chains ofDIA-SUA, DIA-SUA-DIA, DIA-SUA-DIA-SUA, and DIA-SUA-DIA-SUA-DIA moleculescovalently bound to each other, with the two extremities of each bridgeforming amine bonds with free moieties on the tissues.

EXAMPLE 4

A 3-step cross-linking process is employed that is similar to theprocess used in Example 2, but which uses BCA instead of SUA. As such, 7mM BCA was used in the first step, 15 mM DIA in the second step, and 7mM BCA again in the third step. Because BCA has three carboxyls locatedon a benzene ring, the cross-links formed by this process are generallymore complex than the linear bridges obtained when the cross-linkingagent includes an aliphatic chain, such as SUA.

EXAMPLE 5

A cross-linking process is carried out in only two steps, where EDC andsulfo-NHS are used in the presence of DIA in the first step and of SUAin the second step. As a result, three types of links are formed betweenthe free active moieties of the tissue: direct links, one-link bridgesmade of either SUA or DIA, with the two ends of each bridge binding totwo amines or to two carboxyls of the tissue, respectively, and two-linkbridges made of the two cross-linking agents covalently bound to eachother, with one end binding to a free amine of the tissue, and the otherend binding to a free carboxyl group of the tissue.

Fresh porcine valves were incubated first for 48 hours in HEPES buffercontaining 15 mM DIA, and then for 48 hours in HEPES buffer containing10 mM SUA, with 20 mM EDC and 1 mM sulfo-NHS added at each step, withthe valves being rinsed three times with HEPES buffer between the firstand second steps. At the end of the process, cusps and 1×1 wall couponswere dissected from the fixed valves, and stored at room temperature inHEPES buffer containing 20% of isopropanol, pH 7.4. Fresh andglutaraldehyde controls were prepared as described in Example 1.

EXAMPLE 6

An alternative, single step, cross-linking process that uses EDC andsulfo-NHS in the presence of, simultaneously, both cross-linking agentsDIA and SUA, thus forming a more complex network of cross-links, i.e.the types of links and bridges as described in Example 2 and in Example3 above.

Fresh porcine aortic valves were incubated at 4° C. in 200 ml per valveof HEPES buffer containing 15 mM of DIA and 10 mM of SUA. After 24hours, the valves were drained and kept at room temperature for 72 hoursin a solution of 20 mM EDC and 1 mM sulfo-NHS in HEPES buffer. At theend of the process, cusps and 1×1 wall coupons were dissected from thefixed valves, and stored at room temperature in HEPES buffer containing20% of isopropanol, pH 7.4. Glutaraldehyde controls were prepared asdescribed in Example 1.

CHARACTERIZATION OF CROSS-LINKED PORCINE AORTIC VALVES

The porcine aortic valve tissues fixed as described in Examples 1 to 6,inclusive, were subjected to a variety of tests well known to thoseskilled in the art, which determine the degree to which bioprosthetictissues are fixed and cross-linked, and to which they resistcalcification. The cross-linked tissues were also submitted to histologyand biocompatibility studies. Appropriate fresh and glutaraldehyde-fixedsamples, that were prepared and stored as described in Examples 1 to 6,were used as controls.

1. DENATURATION TEMPERATURE

This test is used to evaluate the stability of the cross-linked triplehelical structure of collagen, a major constituent of many bioprosthetictissues, and it consists of recording the temperature at which a samplestarts shrinking when immersed in distilled water placed over a heatsource, this temperature being known to those skilled in the art as theshrink temperature or denaturation temperature. The shrink temperatureincreases as a function of collagen cross-linking. Typically, a sampleis immersed in distilled water at 45° C. The temperature of the water isthen raised at a rate of 1.5° C. per minute until the sample startsshrinking, at which time the temperature is recorded as the denaturationtemperature. The results of this test, based on 3 leaflets from Examples1-6 and the appropriate controls, are reported in Table 1 anddemonstrate that the shrink temperatures are significantly higher forall fixed leaflets than for the fresh leaflets, with the shrinktemperatures for Examples 1 to 6 at least as high as for theglutaraldehyde controls, thus indicating that the collagen cross-linkedas described in Examples 1 to 6 is at least as stable as the collagencross-linked with the standard glutaraldehyde fixation process.

                  TABLE 1                                                         ______________________________________                                                                 SHRINK                                                        CROSS-LINKING AGENTS                                                                          TEMPERATURE                                          EXAMPLES   Step 1    Step 2  Step 3                                                                              Mean ± SEM °C.                   ______________________________________                                        1          none      none    none  86.5 ± 0.2*                             2          SUA       DIA     SUA   88.4 ± 0.2*                             3          DIA       SUA     DIA   90.7 ± 0.1*                             4          BCA       DIA     BCA   88.8 ± 0.0*                             Fresh                              70.3 ± 0.1                              Glutaraldehyde-                    85.4 ± 0.1*                             fixed                                                                         5          SUA       DIA      --   87.1 ± 0.1*                             Fresh                              66.5 ± 0.3                              Glutaraldehyde-                    86.3 ± 0.2*                             fixed                                                                         6          DIA + SUA  --      --   87.4 ± 0.3                              Glutaraldehyde-                    86.5 ± 0.2                              fixed                                                                         ______________________________________                                         *significantly higher than fresh controls, p < 0.05 (NewmanKeuls test)   

2. RESIDUAL AMINE TEST

This test evaluates the stability of the cross-linked tissue bydetermining the number of amine groups that remain free in thebioprosthetic tissue at the end of the cross-linking process. Itconsists of incubating the cross-linked sample in a ninhydrin solution,which alters its coloration in the presence of free amines. Typically,the samples are individually incubated at 95° C. for 20 minutes in 1 mlof ninhydrin in citrate buffer, pH 5.0, dried and weighed. Eachincubation solution is then diluted with 1 ml of 50% isopropanol indistilled water, and its optical density, which is read at 570 nm usinga spectrophotometer, is applied to a standard linear equation determinedby using various concentrations of 1-norleucine, and divided by the dryweight of the sample, thus providing a value of residual aminesexpressed as nanomoles of amines per mg of dry tissue. The results,obtained from 3 leaflets and 3 wall coupons from each of Examples 1 to4, are reported in Table

                  TABLE 2                                                         ______________________________________                                                 CROSS-LINKING                                                                            RESIDUAL AMINES                                                    AGENTS     nmoles/mg dry tissue                                               Step Step   Step   Mean ± SEM                                     EXAMPLES   1      2      3    Leaflets Walls                                  ______________________________________                                        1          none   none   none 50.7 ± 3.3*                                                                         30.3 ± 0.7*                         2          SUA    DIA    SUA  11.0 ± 0.0*                                                                         15.0 ± 1.5*                         3          DIA    SUA    DIA  35.3 ± 1.8*                                                                         24.7 ± 0.7*                         4          BCA    DIA    BCA  18.7 ± 1.8*                                                                         19.7 ± 0.9*                         Fresh                         136.0 ± 2.0                                                                         83.0 ± 7.4                          Glutaraldehyde-                9.3 ± 0.7*                                                                          3.7 ± 0.3*                         fixed                                                                         ______________________________________                                         *Significantly lower than Fresh controls, p < 0.05 (NewmanKeuls test).   

Although a proportion of the free amines expressed for Examples 2 to 4may be explained by unreacted amines of DIA molecules that were anchoredby one extremity only, the tissue cross-linked as in Examples 1 to 4contained significantly less amines than the Fresh controls. Inaddition, significantly more amines remained free when the leaflets andthe walls were fixed in the absence of (Example 1), rather than in thepresence of cross-linking agents (Examples 2 to 4), probably because therelative spatial isolation of many reactive tissue amines permittedconnection to carboxyls on the tissue only by bridges formed by thecross-linking agents. Thus, although fixation in the absence ofcross-linking agents, as described in Example 1, may be adequate fortissues where many free amines are close enough to reactive carboxyls topermit direct amide linking in the presence of a coupling agent, somebioprosthetic tissues, e.g. heart valves, are preferably stabilized inthe presence of one or more cross-linking agent(s).

3. TEST OF RESISTANCE TO COLLAGENASE

This test determines the degree of fixation of bioprosthetic tissues byevaluating their resistance to digestion by collagenase, a proteolyticenzyme specific for collagen, and consists of determining the amount ofamines that are released from tissue when it is incubated in a solutioncontaining collagenase. Resistance to collagenase digestion for 3leaflets and 3 pieces of walls, cross-linked as in Examples 1-6 (withtheir appropriate fresh and glutaraldehyde-fixed controls), was testedby mincing and then incubating each sample at 37° C. for 27 hours in 3ml of a solution containing 5 mg of collagenase, 180 mg of CaCl₂ ·2H₂ Oin HEPES buffer, pH 7.4. The level of amines in each sample wasdetermined by the ninhydrin test previously described (refer to residualamines test), using 0.1 ml of collagenase solution in 1 ml of ninhydrinsolution.

The results, which are reported in Table 3, clearly demonstrate that thelevel of amines released in the collagenase solution is significantlylower for all fixed leaflets and walls than for the Fresh controls, thusindicating that all bioprosthetic tissues prepared as described inExamples 1-6 strongly resist collagenase digestion and are well fixed.

                  TABLE 3                                                         ______________________________________                                        CROSS-LINKING                                                                 AGENTS             AMINES RELEASED                                            EXAM-  Step      Step   Step Mean ± SEM nmoles/mg                          PLES   1         2      3    Leaflets Walls                                   ______________________________________                                        1      none      none   none 5.5 ± 0.5*                                                                          6.4 ± 0.1*                           2      SUA       DIA    SUA  3.9 ± 0.2*                                                                          4.9 ± 0.1*                           3      DIA       SUA    DIA  2.9 ± 0.5*                                                                          5.3 ± 0.4*                           4      BCA       DIA    BCA  4.5 ± 0.8*                                                                          5.5 ± 0.3*                           Fresh                        359.2 ± 22.8                                                                        347.0 ± 24.0                         Glutar-                      0.9 ± 0.1*                                                                          1.0 ± 0.2*                           aldehyde-                                                                     fixed                                                                         5      SUA       DIA     --  16.1 ± 0.6*.sup.#                                                                   17.2 ± 0.5*.sup.#                    Fresh                        .sup. 2260 ± 153.7.sup.#                                                            936.9 ± 76.3.sup.#                   Glutar-                       6.1 ± 1.0*.sup.#                                                                    2.3 ± 0.2*.sup.#                    aldehyde-                                                                     fixed                                                                         6      DIA + SUA  --     --  969.0 ± 7.4*.sup.#                                                                  38.7 ± 4.2*.sup.#                    Glutar-                      9.3 ± 3.6.sup.#                                                                     3.9 ± 3.2.sup.#                      aldehyde-                                                                     fixed                                                                         ______________________________________                                         *Significantly lower than Fresh controls, p < 0.05 (NewmanKeuls test)         .sup.# The samples were incubated for 72 instead of 27 hours.            

The results for Examples 1 to 6 are slightly higher than those for theirrespective glutaraldehyde-fixed controls. This difference is notconsidered to reflect differences in resistance to collagenase butinstead to result from the constant release during incubation ofglutaraldehyde from the glutaraldehyde-fixed samples--which then bindsboth to the collagenase of the solution, thus decreasing the efficacy ofthe collagenase solution, and also to the amines released from thetissue, thus decreasing the number of free amines.

4. TEST OF RESISTANCE TO PROTEASE

This test determines the degree of fixation of bioprosthetic tissues byevaluating their resistance to digestion by protease, a non-specificproteolytic enzyme; it typically consists of determining the weight lostby a tissue that is incubated in a solution containing protease. Thetest was conducted on 3 leaflets and 3 pieces of walls cross-linked asin Examples 1-6 (with their appropriate fresh and glutaraldehyde-fixedcontrols). The samples were blotted, weighed, incubated in 3 ml of asolution prepared by dissolving 75 mg of protease and 75 mg of CaCl₂ ·H₂O in 150 ml of HEPES buffer, pH 7.4,blotted and weighed. The results(expressed as % of weight remaining in the tissue), which are reportedin Table 4, clearly demonstrate that porcine aortic valves prepared asdescribed in Examples 1 to 6 strongly resist non-specific degradation byprotease.

                  TABLE 4                                                         ______________________________________                                                                 % WEIGHT REMAINING                                   EXAM-  CROSS-LINKING AGENTS                                                                            Mean ± SEM                                        PLES   Step 1    Step 2  Step 3                                                                              Leaflets                                                                              Walls                                  ______________________________________                                        1      none      none    none  47.7 ± 0.7*                                                                        50.6 ± 2.2*                         2      SUA       DIA     SUA   59.1 ± 1.7*                                                                        51.8 ± 3.5*                         3      DIA       SUA     DIA   49.6 ± 2.6*                                                                        55.8 ± 1.6*                         4      BCA       DIA     BCA   52.5 ± 3.5*                                                                        49.5 ± 1.0*                         Fresh                          0.0 ± 0.0                                                                          0.8 ± 0.4                           Glutar-                        27.2 ± 1.5*                                                                        32.5 ± 3.2*                         aldehyde-                                                                     fixed                                                                         5      SUA       DIA      --   64.4 ± 4.2*                                                                        40.0 ± 0.5*                         Fresh                          0.0 ± 0.0                                                                          0.0 ± 0.0                           Glutar-                        68.8 ± 4.1*                                                                        34.1 ± 3.2*                         aldehyde-                                                                     fixed                                                                         6      DIA + SUA  --      --   69.9 ± 1.3                                                                         30.4 ± 1.1                          Glutar-                        71.5 ± 3.4                                                                         26.1 ± 0.4                          aldehyde-                                                                     fixed                                                                         ______________________________________                                         *significantly higher than Fresh controls at p < 0.05 (NewmanKeuls test) 

Based on the results of the denaturation temperature test, the residualamines test, and the resistance to collagenase and to protease tests, itis shown that bioprosthetic tissues cross-linked by the processeshereinbefore described which embody various features of the presentinvention are as well fixed and cross-linked as bioprosthetic tissuesfixed with the standard glutaraldehyde process. However, because theamide bonds formed with the present invention are more stable than theSchiff-bases formed with the glutaraldehyde process, the bioprosthetictissues resulting from use of the present invention will not inducelow-grade toxic effects and undergo long-term degeneration, which is aconsiderable advantage over the glutaraldehyde-fixed bioprosthetictissues.

5. TEST OF RESISTANCE TO CALCIFICATION

Another important advantage of the present invention over the standardglutaraldehyde process is illustrated by comparing the calcium levels ofthe tissues cross-linked as described in Examples 1-6 with those ofglutaraldehyde-fixed tissues, when they are implanted subdermally inweaning rats; this is a model of calcification known as a standardscreening model by those who are skilled in the art of bioprostheses.

Six leaflets and 6 wall coupons from porcine aortic valves from each ofExamples 1 to 6, and their appropriate glutaraldehyde-fixed controls,were rinsed 3 times with sterile saline and implanted subdermally for 4weeks or 8 weeks in the abdomen of 3-week old male Sprague-Dawley rats.The retrieved samples were then cleaned of surrounding tissues,lyophilized, weighed, hydrolyzed in 1 ml of ultrapure 6N HCl at 85° C.for 24 hours, and submitted to calcium determination by eitherInductively Coupled Plasma analysis or by Atomic Absorption. The results(Table 5) indicate that the leaflets and the walls of Examples 1-6 weresignificantly less calcified than the glutaraldehyde-fixed controls andthat leaflets were not significantly more calcified at 8 weeks than at 4weeks; they thus demonstrate that porcine heart valves are moreresistant to calcification when cross-linked with the processesembodying features of the present invention than when fixed by thestandard glutaraldehyde process.

                  TABLE 5                                                         ______________________________________                                                          CALCIUM                                                     CROSS-LINKING     mg/g dry sample                                             AGENTS            Mean ± SEM                                               EXAM-   Step    Step   Step Leaflets    Walls                                 PLES    1       2      3    4-week 8-week 4-week                              ______________________________________                                        1       none    none   none  25 ± 15*                                                                          21 ± 19*                                                                          40 ± 8*                         2       SUA     DIA    SUA   9 ± 5*                                                                            31 ± 21*                                                                          35 ± 4*                         3       DIA     SUA    DIA   8 ± 5*                                                                            19 ± 18*                                                                          26 ± 14*                        4       BCA     DIA    BCA   4 ± 2*                                                                            36 ± 13*                                                                          33 ± 5*                         Glutar-                     204 ± 10                                                                          230 ± 34                                                                          130 ± 8                          aldehyde-                                                                     fixed                                                                         5       SUA     DIA          24 ± 6                                                                             --    85 ± 15                         Glutar-                     220 ± 8                                                                             --   105 ± 5                          aldehyde-                                                                     fixed                                                                         6       DIA ±                                                                              --     --    1 ± 1*                                                                             --    43 ± 7*                         Glutar- SUA                 185 ± 11                                                                            --    65 ± 3                          aldehyde-                                                                     fixed                                                                         ______________________________________                                         *Significantly lower than glutaraldehyde controls, p < 0.05 (NewmanKeuls      test)                                                                    

These results with respect to calcification in the rat model show thatthe high rate of failure of bioprosthetic devices, which is believed tobe currently due primarily to calcification, should be considerablyreduced by use of the present invention.

6. HISTOLOGY STUDIES

These studies were performed to ensure that the cross-linking processesdescribed in the present invention do not induce deleterious effects onthe structure of the bioprosthetic tissue that could adversely affectthe function of the bioprosthetic tissue, and they also are effective toevaluate the "quality" of these tissues before and after implantation.

One such study was performed by viewing bioprosthetic tissuescross-linked as described in the present invention under scanningelectron microscopy. Three leaflets from each of Examples 1 to 4 andfrom their appropriate glutaraldehyde-fixed controls were cuttransversely to expose the internal layers (fibrosa, spongiosa andventricularis). They were then critically point-dried in ethanol, coatedwith AuPd and examined using a Hitachi S-800 field emission scanningelectron microscope at 15 KV. This study demonstrated that the leafletsfrom Examples 1 to 4 had normal morphology; the tissue was compact,there was no sign of delamination, and the inflow and outflow surfacesof the leaflets showed no sign of roughening.

The other histology study was performed by viewing, under lightmicroscope, unimplanted and implanted leaflets from porcine aorticvalves that have been cross-linked as described using processesembodying various features of the present invention. Three unimplantedleaflets, and three leaflets subdermally implanted in rats for 4 weeks,from each of Examples 1 to 4 and from their appropriateglutaraldehyde-fixed controls, were placed in 4% glutaraldehyde and sentto Dr. Frederick Schoen, Brigham and Women's Hospital, Boston, Mass.,where they were embedded in JB-4 glycol methacrylate medium. Sections 2to 3 μm thick were then stained for cells with hematoxylin and eosin,for calcium salts with the von Kossa stain, and for collagen, elastinand mucopolysaccharides with the Movat pentachrome stain. Althoughleaflets from Examples 1 (3 steps with EDC and sulfo-NHS in the absenceof cross-linking agents) and 4 (3-step treatment with the cross-linkingBCA used for the first and third steps instead of SUA) occasionallyexhibited mild edema and moderate smudging of the fibrosa collagen, theyappeared much less calcified than the glutaraldehyde-fixed leaflets. Onthe other hand, the leaflets treated in Examples 2 and 3 (3-stepreactions with the cross-linking agents SUA/DIA/SUA and DIA/SUA/DIA,respectively) appeared to be better preserved, and to calcifyconsiderably less, than glutaraldehyde-fixed leaflets, withoutexhibiting any sign of inflammatory reaction.

8. BIOCOMPATIBILITY STUDY

Twenty leaflets from Example 1 were sent to Dr. James A. Anderson atCase Western University, Cleveland, Ohio, where they were sterilized,placed in small stainless steel wire mesh cages and implantedsubdermally in rats. Empty cages served as controls. The degree ofinflammatory response was determined at 4, 7, 14 and 21 days ofimplantation by quantitative and differential measurement of leucocytes,polymorphonuclear and macrophage counts, and by alkaline and acidphosphatase analyses of the exudate that collected in the cages.

The results of these measurements and analyses demonstrated that theleaflets were found to be biocompatible and nontoxic, thus indicatingthat bioprosthetic tissues cross-linked by processes embodying featuresof the present invention are suitable for implantation.

EXAMPLE 7

The processes of Examples 2 and 3 are repeated except that, in bothinstances the fixation processes are halted after the first step. Afterdraining and rinsing, the samples are stored in HEPES buffer at pH 7.4containing 20% isopropanol at room temperature. Testing for thermaldenaturation, collagenase digestion, protease digestion and resistanceto calcification is carried out as reported hereinbefore, together withappropriate control samples of fresh tissue, and the resultantcross-linked materials are compared to glutaraldehyde-treated samples.The samples incubated either with a mixture of diamine, EDC andsulfo-NHS or with a mixture of suberic acid, EDC and sulfo-NHS areconsidered to exhibit thermal stability and resistance to proteasedigestion far superior to fresh tissue and as good as theglutaraldehyde-treated samples. Although the resistance to collagenasedigestion may not be quite as good as glutaraldehyde-treated samples, itis considered to be fully adequate. Resistance to calcification of bothsuch sets of cross-linked samples is considered to be superior to thatof glutaraldehyde-fixed material.

Although the invention has been described with regard to a number ofpreferred embodiments, which constitute the best mode presently known tothe inventors for carrying out this invention, it should be understoodthat various changes and modifications, as would be obvious to onehaving the ordinary skill in this art, may be made without departingfrom the scope of the invention which is defined by the claims that areappended hereto. For example, if the initial treatment of a 3-stepprocess using cross-linking agents at each step is carried out using onedicarboxylic acid, and although it may be preferable to employ the sametype of carboxylic acid for the third step of the reaction, a differentdicarboxylic acid or a tricarboxylic acid could be alternativelyemployed. The foregoing similarly applies when a diamine is employed inthe first treatment step. Rather than using a single solution containingboth the coupling agent and the cross-linking agent, treatment may becarried out sequentially with two separate solutions. Also, although 2or 3 steps may be preferred to provide adequate cross-linking for sometissues, other tissues may already be adequately cross-linked after asingle step.

Particular features of the invention are emphasized in the claims thatfollow.

What is claimed is:
 1. A process for fixing animal tissue to render itsuitable for implantation in living mammals, comprisingtreating saidanimal tissue with an effective amount of a coupling agent whichpromotes the formation of amide bonds between reactive carboxy moietiesand reactive amino moieties in combination with a coupling enhancer, soas to result in the formation of amidated links to reactive moietiescarried by the molecules of said animal tissue to render said tissueresistant to protease digestion and to calcification.
 2. The process ofclaim 1 wherein said tissue is also treated with a cross-linking agentcontaining either at least two reactive amine moieties or at least tworeactive carboxy moieties.
 3. The process of claim 2 wherein saidcross-linking agent is a water-soluble di- or tri-amine or awater-soluble di- or tri-carboxylic acid, and said coupling agent iswater-soluble.
 4. The process of claim 3 wherein said cross-linkingagent has a carbon chain at least 4 carbon atoms in length.
 5. Theprocess of claim 1 wherein said coupling agent is 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC).
 6. A prosthesis treated according tothe process of claim 1 which resists calcification.
 7. A prosthesistreated according to the process of claim 5 which resists calcification.8. A process according to claim 1 comprising the steps of:a) treatingsaid animal tissue with a first cross-linking agent containing either atleast two reactive amino groups or at least two reactive carboxylgroups, in the presence of said coupling agent, such that at least oneof said reactive groups forms an amide bond with a reactive moiety onsaid tissue molecules while another reactive group on at least someportion of said first cross-linking agent remains free; and b) repeatingthe treatment described in (a) in the presence of said coupling agentwith a second cross-linking agent containing at least two reactivecarboxyl groups if said first cross-linking agent used in (a) containsamino groups, or vice-versa if said first cross-linking agent containscarboxyl groups, such that additional amide bonds are formed betweenreactive groups of said second cross-linking agent and either said freegroups on said first cross-linking agent or reactive moieties on saidtissue molecules, resulting in the formation of links between or withinthe molecules of said animal tissue wherein some of said links arechains containing at least one of both said first and secondcross-linking agents.
 9. The process of claim 8 wherein step (a) isrepeated after step (b) using a third cross-linking agent which is thesame as or equivalent to said first cross-linking agent so as to createfurther amide bonds (i) between one reactive group of said thirdcross-linking agent and a free reactive group on said secondcross-linking agent and (ii) between another reactive group on saidthird cross-linking agent and a free reactive moiety on a tissuemolecule, thereby increasing the number of said links formed between andwithin the molecules of said tissue.
 10. The process of claim 8 whereinsaid first and second cross-linking agent comprise water-soluble di- ortri-amines and water-soluble di- or tri-carboxylic acids and saidcoupling agent is water-soluble.
 11. The process of claim 8 wherein saidfirst and second cross-linking agents are each at least 4 carbon atomsin length.
 12. The process of claim 8 wherein step (a) employs 1,6hexane diamine, and step (b) employs suberic acid or1,3,5-benzenetricarboxylic acid.
 13. The process of claim 8 wherein step(a) employs suberic acid or 1,3,5-benzenetricarboxylic acid and step (b)employs 1,6 hexane diamine.
 14. A prosthesis treated according to theprocess of claim
 8. 15. A process for fixing animal tissue to render itsuitable for implantation in living mammals, which processcomprisestreating said animal tissue with an aqueous solution whichcontains a water-soluble first reagent having at least 2 reactive aminegroups, and a water-soluble second reagent containing at least 2reactive carboxyl groups, and also with a water-soluble coupling agentplus a water-soluble coupling enhancer, such that said reactive amineand carboxylic groups are promoted to form amide bonds with tissuemolecules having reactive moieties thereon, and washing said treatedanimal tissue to remove unreacted reagents and render it suitable forimplantation.
 16. (Amended) A process according to claim 15 wherein saidanimal tissue is sequentially treated with said tissue being firstcontacted with a first aqueous solution containing said first and secondreagents and after removal of said first solution being thereaftertreated with a second aqueous solution containing said water-solublecoupling agent plus said water-soluble coupling enhancer that promotessaid reactive groups to form amide bonds.
 17. A process according toclaim 16 wherein said second solution contains 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide(sulfo-NHS) as said water-soluble coupling agent and said water-solublecoupling enhancer.
 18. A prosthesis formed at least partially ofprosthetic tissue containing cross-links between and within theproteinaceous molecules of said prosthetic tissue, which cross-links arecomprised of amide bonds between reactive moieties on said tissue andadditional amide bonds between reactive moieties on said tissue andfirst and second cross-linking agents, both of which are between 4carbon atoms and 8 carbon atoms in length and which first cross-linkingagents have at least 2 reactive amino groups and which secondcross-linking agents have at least 2 reactive carboxyl groups, whichcross-linking of said prosthetic tissue via said amide bonds and saidadditional amide bonds is such that the prosthetic tissue is fixed,resists calcification and does not induce inflammatory responses uponimplantation into a living mammal.
 19. The prosthesis of claim 18wherein said cross-links which include said additional amide bonds areformed of residues of said cross-linking agents which are selected fromthe group consisting of 1,6 hexane diamine, suberic acid and1,3,5-benzenetri carboxylic acid.